Abstract
The rat Na(+)- and Cl(-)-dependent serotonin transporter was expressed in Sf9 insect cells using the baculovirus system. Expression of the serotonin transporter caused the Sf9 cells to accumulate [3H]serotonin (Km 78 nM) and to bind the specific transport inhibitor [125I]RT155 (2 beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane) (Kd 0.22 nM). Ligand binding assays on isolated membranes showed 500,000 copies of the serotonin transporter/cell (9 pmol/mg of membrane protein). Immunoreactive bands of apparent M(r) 54,000 (unglycosylated) and 60,000 (glycosylated) were observed in Western blots of membrane proteins from infected cells. The 54-kDa band was significantly smaller than the expected M(r) of 72,500 predicted from the cDNA sequence. The 54-kDa band was shown to represent the intact serotonin transporter by expressing a recombinant serotonin transporter that contained c-Myc and FLAG epitope tags engineered at the N and C termini, respectively. Both tags were present on a membrane protein that migrated slightly slower than the previously observed 54-kDa band, consistent with the extra mass added by the tags. The tags did not affect the Kd for [125I]RT155 binding. The effect of N-linked glycosylation on ligand binding and the level of expression were studied. The expression of the serotonin transporter in tunicamycin-treated Sf9 cells resulted in low levels of ligand binding activity (0.2 pmol/mg) but unchanged Kd. Similarly, mutated serotonin transporters that contained reduced numbers of N-linked glycosylation sites had unchanged Kd for [125I]RT155 binding whether there were 2, 1, or 0 N-linked glycosylation sites present on the serotonin transporter. In contrast, Bmax was dramatically reduced; levels of expression of the unglycosylated serotonin transporter (0.4 pmol/mg) were 20-fold lower compared with levels of the fully glycosylated serotonin transporter. The Km for [3H]serotonin uptake was also unchanged. These data indicate that glycosylation is required for optimal stability of the serotonin transporter in the membrane but not for serotonin transport or ligand binding per se.
Highlights
The rat Na+-and C1--dependent serotonin transporter rotonin into the cell.The serotonin transwas expressed in Sf9 insect cells using the baculovirus porter isalso present in peripheral tissues sucahs platelets [2]
The binding of [1251]RT155to cDNA sequence (72,500,see below). Both anti-serotonin trans- the expressed serotonintransporter occurred with high affinity porter antibodies were specific for the serotonin transporter (Table I); no binding was observed in cells infected with theconbecause they did not bind to any other membrane proteins in trol recombinant baculovirusbv-pVL(Fig. 4)
Expression of the serotonin transporter in a heterologous system allows the manipulation of the cDNA sequence to include affinity tags, such as a hexahistidine tail, that can be used as anefficient purification step [35, 36]
Summary
Rather thansequence the whole of the rSERT gene to ensure no other mutations were introduced during the mutagenesis reaction, a n RsrIIIAZwNI fragment that contained the mutated cAodsonn was gel purifiedfromplasmid pCGT133 andligatedinto AZwNI(partia1)l the rat serotonin transporter inSf9cells using thebaculovirus RsrII-digested plasmid pCGTlO9 t o create plasmid pCGT142 (see Fig. system and the effect of N-linked glycosylation on serotonin Id).The DNA sequence at the restrictionsiteswassubsequently transport and E'251]RT155 ligand binding. CMYC2) encoding the c-Myc tag were synthesized so that when the baculovirus stocks was performedas described by Summers and Smith oligonucleotides were annealed there were NcoI-compatible overhangs at both ends These were ligated directly into NcoI-digested plasmid pCGT109, and the orientation of the insert was determined by DNA.
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